Project Summary The control of DNA topology has a major impact on the flow of genetic information. The present application focuses on type II topoisomerases, molecular machines that modulate DNA supercoiling and remove chromosome entanglements by catalyzing the ATP-dependent transport of one DNA duplex through another. Type II topoisomerases are critical for maintaining gene expression, chromosome superstructure, and genome integrity; they also serve as frontline drug targets for treating infectious disease and cancer. Progress during the prior project period, along with new data, has revealed an unanticipated richness to topoisomerase diversity and action, setting the stage for an integrated set of three aims that tackle previously unapproachable issues of how topoisomerases operate and how they can be controlled by therapeutics and biological effectors. Aim 1 will address how ATP drives the chemomechanical action and kinetic timing of type II topoisomerases, an issue central to the function of molecular machines as a whole. Aim 2 will establish how quinolones, one of the most successful antibiotic classes, discriminate between bacterial and human type II topoisomerases; the resulting molecular understanding of drug-protein interactions will form a framework for improving therapeutic selectivity. Aim 3 will break new ground in the emerging and poorly- understood area of topoisomerase regulation, mechanistically defining how an prototypical representative of three different classes of effectors - natural small molecules, post-translationa modifications, and protein-protein interactions - activate or attenuate topoisomerase function. Our methodological approach is distinguished by a comprehensive blend of biochemical, structural, genetic, and single-molecule methodologies in which developments for one aim promote work on the others. Past progress and unpublished findings establish feasibility for the proposed effort. Our studies are expected to impact multiple fields, from the study of molecular machines and the control of DNA dynamics, to understanding how cells and anti-topoisomerase agents alter topoisomerase activity to support specific physiological needs and promote human health.